Home Cryostructuring of polymer systems. 47. Preparation of wide porous gelatin-based cryostructurates in sterilizing organic media and assessment of the suitability of thus formed matrices as spongy scaffolds for 3D cell culturing
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Cryostructuring of polymer systems. 47. Preparation of wide porous gelatin-based cryostructurates in sterilizing organic media and assessment of the suitability of thus formed matrices as spongy scaffolds for 3D cell culturing

  • Vladimir I. Lozinsky EMAIL logo , Valentina K. Kulakova , Roman V. Ivanov , Alexander Yu. Petrenko , Olena Yu. Rogulska and Yuriy A. Petrenko
Published/Copyright: September 22, 2017
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e-Polymers
From the journal e-Polymers Volume 18 Issue 2

Abstract

New gelatin-based cryostructurates have been elaborated and tested as scaffolds for three-dimensional (3D) cell culturing. Scaffold preparation included dissolution of Type A gelatin in dimethylsulfoxide, freezing of such solution, cryoextraction of crystalline phase with cold ethanol, cross-linking of gelatin with carbodiimide in ethanol medium, treatment of the matrix with ethanolic solution of Tris and tanning of the matrix with formaldehyde dissolved in ethanol. The use of organic media during all the preparation stages ensured the sterility of the scaffolds. The matrices thus prepared were seeded with human adipose tissue multipotent mesenchymal stromal cells to confirm the biocompatibility of scaffolds and their possibility to provide necessary environment for the cell growth and differentiation. The cells attached onto the surface of the pore walls, proliferated and differentiated into osteogenic and adipogenic lineages. These results demonstrate that gelatin-based cryostructurates prepared in the sterility ensuring organic media can be used as scaffolds for tissue engineering purposes.

Keywords: 3D culturing-differentiation; mesenchymal stromal cells; spongy scaffold; type A gelatin

1 Introduction

Gelatin is well known to be a biopolymer derived from animal collagen which, in turn, is the most prevalent protein in the extracellular matrix (ECM) and is the dominant component in human soft and hard connective tissues (1), (2), (3). The chemical composition of gelatin is close to collagen, but lacks the antigenicity and the immunogenicity (4), (5). As a mimic component of ECM, gelatin provides an ideal environment for cell attachment and growth. This feature makes gelatin an attractive material for the development of scaffolds for tissue engineering (6), (7), (8), (9), (10), (11), as well as for the expansion of mesenchymal stromal cells (MSCs) (12). In the latter case, due to the capacity to multilineage differentiation, MSCs are widely used as a cellular component of the tissue-engineered constructs. Among the sources of the MSCs, human adipose tissue has the advantage of being a discard product, obtained mainly via low invasive liposuction. The MSCs derived from human adipose tissue have been successfully differentiated into bone, cartilage, fat (13), (14) and endothelial cells (15).

Scaffolds for tissue engineering applications should provide the necessary environment for optimal growth and differentiation of cells, while being maximally biocompatible and safe. In this context, bacterial contamination of raw gelatin material represents a challenging problem for the application of gelatin-based matrices, as the contamination of a standard gelatin production process with a variety of Gram-positive and Gram-negative bacteria has been demonstrated (16). Even though the extreme temperature and pH conditions were employed during gelatin manufacturing, thermotolerant aerobic endospore-forming bacteria, attributed to the genus Bacillus, could be identified in the semi-final product (17). This necessitates the need for intensive sterilization of gelatin-based materials, prior to application. However, thermal or chemical sterilization techniques are not frequently applicable for the gelatin scaffolds, especially those that contain certain additional biologically active components. It was also shown that γ irradiation resulted in worsening the mechanical properties of gelatin films (18). Moreover, when the γ sterilization was applied to gelatin-chitosan-tricalcium phosphate scaffolds, both a decrease in the pore sizes and an increase of the pore wall thickness of the three-dimensional (3D) structures have been observed (19). Such changes in scaffold properties under the influence of sterilization can vary in regard to initial matrix characteristics, e.g. the gelatin concentration, the presence of additional components or the type of cross-linking procedure. Furthermore, sterilization by γ irradiation is a cost- and labor-intensive technique which additionally increases the cost of gelatin-based materials. Thus, the search for alternative, reproducible and cost-effective methods for fabrication of sterile scaffolds would be advantageous for their wide application in tissue engineering and regenerative medicine.

A further principle requirement for such scaffolds is their macroporosity of an interconnected character in order to ensure the non-hindered cells penetration in the matrix bulk and their adhesion to the inner surfaces of pore walls (20), (21), (22), (23), (24). In this context, several approaches have been developed for the preparation of macroporous matrices (20), (25), (26), and among such approaches, those based on cryogenic structuring, particularly cryotropic gel-formation (27), (28), (29), (30) and freeze-drying (31), (32), (33), became very popular in recent years for the creation of gelatin alone or gelatin-containing macroporous materials (11), (20), (21), (22), (23), (22), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53), (54), (55), (56).

As for the polymeric matrices that are formed as a result of cryogenic procedures employed for the fabrication of such macroporous polymeric scaffolds, there are two their types: cryogels (27), (28), (29), (30), (57), (58), (59), when the buildup of the 3D polymeric network and its simultaneous cross-linking via chemical or physical bonds occur within the moderately-frozen system (Figure 1A), and cryostructurates (27), (43), (50), (58), (60), when the feed solution of polymeric precursors is initially frozen, the formed solvent crystals (ice in the case of aqueous media) are further removed via sublimation or cryoextraction techniques. It results in obtaining a macroporous “foam”, and only then the cryogenically-structured matrix is chemically fixed by stable interchain bonds using suitable cross-linking methods (Figure 1B). This latter approach allows preparing wide-pore materials originating from different synthetic and natural polymers. The pore sizes and porous “architecture” of the resultant cryostructurates depend on the chemical nature of the precursors, their molecular-weight characteristics, concentration in the initial solution, the type of used solvent and freezing conditions. In doing so, if it is possible to perform the cryostructuring process in an organic solvent capable of killing various microorganisms, high sterility of the final material can be ensured.

Figure 1: General scheme showing the differences in the pathways for producing of the cryogel-type polymeric matrices (A) and the cryostructurate-type matrices (B).Dashed vertical line separates the pathways for the preparation of these cryogenically structured polymeric materials.
Figure 1:

General scheme showing the differences in the pathways for producing of the cryogel-type polymeric matrices (A) and the cryostructurate-type matrices (B).

Dashed vertical line separates the pathways for the preparation of these cryogenically structured polymeric materials.

Based on the above-discussed considerations, we have generated the idea of this study which aim was to prepare biocompatible and sterile wide porous gelatin matrices and to evaluate their potential of performing as scaffolds for the 3D cell culture, human adipose tissue multipotent MSCs in this particular case. To the best of our knowledge, there were no previous reports on the preparation of gelatin scaffolds via the cryostructurate-based pathway (Figure 1B) using the cryoextraction technique and, moreover, performing the processing procedures exactly in the non-aqueous media.

2 Materials and methods

2.1 Materials

The following substances and reagents were used as received: Gelatin from porcine skin, Type A, 300 Bloom (GA), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide) (EDC), Minimal Essential Medium-α modification (α-MEM), L-glutamine, fluorescein diacetate (FDA), ethidium bromide (EB), ascorbic acid, β-glycerophosphate, dexamethasone, 3-isobutyl-1-methyl-xanthine, dexamethasone, insulin, indomethacin, Nile Red dye and Fast Blue RR Salts/Naphtol kit – all from Sigma-Aldrich Inc. (St. Louis, MO, USA); Amido black dye was from Merck GmbH (Darmstadt, Germany); tris(hydroxymethyl)aminomethane (Tris) (high purity grade) was from GERBU Trading GmbH (Gaiberg, Germany); ethanol (95%, medical grade) was from ECOLab (Moscow, Russian Federation), and 40% aqueous formaldehyde solution was from Reakhim (Moscow, Russian Federation); fetal bovine serum (FS), phosphate buffered saline (PBS), penicillin and streptomycin were from Biowest (Nuaille, France); Hank’s solution was from PAA (Pasching, Austria) and Alamar Blue redox indicator (AB) – from Serotec (Bio-Rad, Raleigh, NC, USA). Dimethylsulfoxide (DMSO) (chemically pure grade; Reakhim, Moscow, Russian Federation) was additionally purified by a freezing-out technique. Adhesive polystyrene cell culture flasks and plates were from TPP (Trasadingen, Switzerland). A Quant-iT PicoGreen dsDNA assay kit was from Invitrogen (Eugene, OR, USA); ALP 2×60 and CaL 1×250 kits – both from Biolatest Lachema (Brno, Czech Republic). The monoclonal antibodies used were as follows: CD29-PE and CD45-PE were from Serotec (Bio-Rad, Raleigh, NC, USA), CD90-FITC and CD105-FITC – from Serotec (Kidlington, UK), CD34-FITC – from Dako (Glostrup, Denmark), and CD73-PE – from BD Biosciences (Oxford, UK).

2.2 Methods

2.2.1 Preparation of gelatin cryostructurates

The gelatin-based wide pore cryostructurates were fabricated essentially in accordance with the patented procedure (61). In brief, the necessary amount of gelatin powder was suspended in a known volume of DMSO and then dissolved under stirring at 60°C. After cooling down to 35–40°C the prepared transparent gelatin solution was poured by the 2-mm-layers into 35-mm plastic Petri dishes that were quickly installed on a strictly horizontal cooling metal plate connected to the outer coolant contour of an ultra cryostat circulator LOIP LT-116a (Laboratory Equipment and Instruments Ltd., St. Petersburg, Russian Federation), where the samples were frozen and incubated at −20°C for 2 h. The crystals of frozen DMSO were thereafter extracted with cold ethanol for 3 days at −20°C, the ethanol being daily changed for a fresh portion. The gelatin macromolecules within the matter of pore walls in the porous disks thus obtained were further cross-linked by their incubation in 0.05 m ethanolic solution of EDC at room temperature for 48 h followed by rinsing with pure ethanol. Then the disks were immersed in ethanolic solution of Tris (5 mg ml−1) at room temperature for 18 h and again rinsed with pure ethanol. Finally the samples were additionally tanned by the treatment with 2% (v/v) ethanolic solution of formaldehyde at room temperature for 48 h followed by rinsing with ethanol several times and further, prior to use, stored under the ethanol layer.

2.2.2 Characterization of gelatin-based cryostructurates

The values of total volumetric swelling degree (Stv/w) for the gelatin discs were found gravimetrically as the amount of water absorbed by 1 g of dry polymer after swelling to equilibrium; the following formula was used assuming water density dH2O=1 g ml−1:

[1]Stv/w=(mt–md)​/​dH2O:md(ml H2O per 1 g of dry polymer),

where mt is the total weight of the water-swollen gelatin spongy cryostructurate, md is the weight of the dried sample.

The swelling degree by weight (Sw/w) of the gel phase (the walls of macropores) for the GA-based cryostructurates were measured after pressing out free capillary water from each water-swollen spongy disc between the several layers of filter paper under a load of ca. 50 g. The filters were changed till the absence of a wet spot on the fresh filter; the disc was then weighed and dried up to a constant weight. The swelling degree was calculated with a formula:

[2]Sw/w=(mws–md):md (g H2O per 1 g of dry polymer),

where mws is the weight of the squeezed gelatin sponge after removal of free water, md is the weight of the dried sample.

2.2.3 Microstructure of gelatin-based cryostructurates

The wide-porous morphology of GA-sponge discs was studied using two techniques of optical microscopy: (i) a conventional light transmission method (optical microscope Eclipse 55i (Nikon, Tokyo, Japan) equipped with a system for digitally recording images) and (ii) a laser confocal microscope Zeiss LSM 510 META (Carl Zeiss, Jena, Germany). The images were taken for both flat surfaces of the discs: the lower face which contacted with the mold bottom upon cryostructuring process, and the top face being in contact with air. In the case of (i), the water-discs, prior to exploration, were stained by treatment with 0.1 mm aqueous solution of Amido Black dye for 1 min followed by rinsing with water. In the case of (ii) the specimens were stained with 0.125 mm solution of Methylene Blue dye.

2.2.4 Cell isolation and culture

The MSCs were isolated from the lipoaspirate of adult patients, after receiving the written consent of the informed healthy volunteer donors, in accordance with the recommendations of the World Medical Association Declaration of Helsinki. The MSCs were obtained by collagenase digestion according to the previously described method (13).

The MSCs were cultured in T75 adhesive polystyrene cell culture flasks at 37°C, 5% CO2, and 95% humidity in α-MEM containing 10% FS, 50 μg ml−1 penicillin, 50 μg ml−1 streptomycin and 0.2 mm L-glutamine. The complete medium changes were performed every 3–4 days. On reaching 80% confluence, the cells were trypsinized, counted with a hemocytometer, and subcultured using a 1:5 plating ratio. The MSCs at passages from 4 to 6 were used for the experiments described below.

2.2.5 3D bio-construct preparation

Spongy gelatin matrices were cut manually to a size of 5×5×2 mm and stored in 70% aqueous ethanol at 4°C. Before seeding, the scaffolds were carefully rinsed with Hank’s solution and placed into the culture medium for 15 min. After culture medium removal, the scaffolds were seeded with the MSCs (3×105 cells/scaffold) using a perfusion technique (62). The bio-constructs obtained were incubated for 2 h to allow cell adhesion and then transferred into the wells of a 24-well adhesive culture plate, containing complete culture medium.

2.2.6 Morphology and metabolic activity of MSCs in scaffolds

Following 7 days of culture, morphology and membrane integrity of the MSCs within the scaffolds were estimated using a double fluorescent staining with FDA and EB as previously described (63). After incubation and rinsing, FDA/EB staining was analyzed by confocal microscope Zeiss LSM 510 META (Carl Zeiss, Jena, Germany). Confocal images were obtained along the Z-axis with 20 μm intervals at an excitation wavelength of 488 nm for FDA and 543 nm for EB.

Metabolic activity of the MSCs in 3D scaffolds on the 1st, 4th and 7th day of 3D culture was assessed by an QUANT Alamar Blue redox indicator. Before analysis 3D constructs were transferred in a new 24-well plate and incubated for 3 h with culture medium containing 10% AB. Reduced AB solution was collected and the fluorescence level of AB was assessed by TECAN GENios microplate reader (Tecan Genios, Grödig, Austria) with an excitation wavelength of 550 nm and an emission wavelength of 590 nm. The difference in fluorescence between the experimental (matrices with the cells) and negative control samples (matrices without the cells) was used as AB value (n=5) and expressed in relative fluorescence units (RFU).

2.2.7 DNA quantification

The DNA content of MSCs cultured onto gelatin scaffolds was evaluated using a Quant-iT PicoGreen dsDNA assay. Samples were collected after 1 or 7 day of culture, washed with PBS and transferred into Eppendorf tubes containing 1 ml of sterile ultra-pure water and then stored in a freezer at −80°C. Before assay, the constructs were thawed at room temperature and sonicated for 15 min to ensure complete cell lysis. The procedure followed was based on the manufacturer’s instructions. Fluorescence intensity was quantified using a TECAN GENios microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

2.2.8 Osteogenic differentiation

Osteogenic differentiation medium consisted of α-MEM, supplemented with 10% FS, 50 μg/ml penicillin, 50 μg ml−1 streptomycin and 0.2 mm L-glutamine with osteogenic inducers: 0.2 mm ascorbic acid, 10 mm β-glycerophosphate, and 1 μm dexamethasone. The corresponding control samples were cultured in complete culture medium without osteogenic supplements. The complete medium changes were performed every 3–4 days. After 21 days of culture, the constructs were washed with PBS, fixed in 4% buffered formalin at 4°C for 30 min. Each scaffold sample was assessed for expression of alkaline phosphatase (ALP) using a fast blue RR salts/naphtol kit according to the manufacturer’s instructions.

2.2.9 Adipogenic differentiation

Adipogenic differentiation medium consisted of α-MEM, supplemented with 10% FS, 50 μg ml−1 penicillin, 50 μg ml−1 streptomycin, and 0.2 mm L-glutamine, and the following adipogenic stimulants: 0.5 mm 3-isobutyl-1-methyl-xanthine, 1 μm dexamethasone, 10 μg ml−1 insulin, and 100 μm indomethacin. The corresponding control samples were cultured in complete culture medium without adipogenic supplements. The complete medium changes were performed every 3–4 days. After 21 days of culture with adipogenic supplements, cells were washed with PBS, fixed in 4% buffered formalin at 4°C for 30 min and stained with Nile Red (1 μg ml−1 in phosphate buffered saline) solution according to the manufacturer’s instructions.

2.2.10 Statistical analysis

The data were evaluated using a nonparametric Mann-Whitney U-test and expressed as mean±SD with n indicating the number of independent experiments. Differences were considered significant with p<0.05. Statistical analyses were performed using the Past version 3.0 software package (64).

3 Results and discussion

3.1 Preparation of gelatin-based cryostructurates

It is commonly known that gelatin is produced by the physical/chemical denaturation of the natural fibrillar protein – collagen (1), (2), (3). There are two major techniques for the transformation of raw collagen materials to gelatin: so-called “acidic” and “basic” procedures whose final products are, therefore, termed as the “acidic” or Type A and the “basic” or Type B gelatins, respectively. Since in the course of collagen processing under the alkaline conditions partial saponification of amide groups in asparaginyl and glutaminyl units of the protein occurs, the isoelectric point of the resultant gelatin is shifted to the acid zone from pI≈8–9 inherent in natural collagens to pI≈5 characteristic of Type B gelatins, whereas in the case of the “acidic” technique such amides are mainly remained intact. Thus, the isoelectric point of Type A gelatins does not change significantly in comparison with collagen. That is why, and taking into account high affinity of numerous anchorage-dependent cell lines to collagen, Type A gelatin was used in this work as a precursor polymer for the preparation of gelatin-based scaffolds. The principal scheme of such matrices fabrication is given by the diagram in Figure 2.

Figure 2: Schematic diagram of the preparation of gelatin-based wide pore crystructurates.
Figure 2:

Schematic diagram of the preparation of gelatin-based wide pore crystructurates.

This scheme includes six major steps, whose application was stipulated by the following considerations:

First, all the stages (i–vi) were performed in the sterilizing organic media, using DMSO (including heating at the stage (i) upon the gelatin dissolution) and ethanol, thus ensuring the maintenance of highly sterile conditions during manipulations in an ordinary chemical laboratory. It is also necessary to point out that neat DMSO efficiently destroys membranes of microbial cell and results in their death (65).

Second, gelatin used in this study was well-soluble in DMSO. Therefore, it was possible to obtain rather high concentrated solutions of this polymer (at least, 10–15 wt.%). Aqueous solutions of the same gelatin concentrations are gelled quickly at 35–30°C, upon cooling, the already formed gels freeze rather than the solutions. This sequence of processes interferes with the effective liquid-solid phase separation during solvent crystallization. On the contrary, the GA-solutions in DMSO were not gelling upon cooling to the temperatures, where the system started to freeze at the stage (ii).

Third, as the crystallization/melting point of DMSO is about +18.5°C (66) its solutions can freeze even at reduced positive (in the centigrade scale) temperatures. However, our preliminary experiments showed that the most reproducible results with respect to final cryostructurates preparation were obtained when the GA concentration in its initial DMSO solutions lay in the range of 7–10% (w/v), and the samples at the stage (ii) were frozen at temperatures from −15 to −25°C. In the current study the gelatin-based scaffolds were prepared by freezing of the feed GA-DMSO-solutions at −20°C.

Fourth, the cryoextraction of frozen DMSO crystalline matter at stage (iii) was performed by cold (−20°C) ethanol which was the solvent for DMSO, but not for gelatin. As such diffusion-rate-dependent process was a rather slow, it required a relatively prolonged time (~3 days for discs of 2-mm-thickness) to ensure the integrity of the gelatin sponge so long as chemical tanning was accomplished.

Fifth, subsequent cross-linking of gelatin macromolecules within the thin pore walls was realized at the stage (iv) using EDC dissolved in ethanol as the same approach we recently employed successfully for a similar “solid-phase” cross-linking of serum-albumin-based cryostructurates (60). Because this carbodiimide reagent was in a large excess relatively to the carboxy- and amino-groups of GA, it was then necessary to deactivate the unreacted pendant O-acylisourea residues. This was done at the stage (v) by the incubation of cross-linked gelatin discs in an ethanolic solution of Tris.

Finally, the discs were additionally tanned by formalin similar to the collagen-containing medical xenografts like skin or cornea (67).

3.2 Swelling characteristics and microstructure of gelatin cryostructurates

When storing in ethanol medium, the GA-based discs prepared accordingly to the scheme shown in Figure 1 were the turbid white-color semi-solid materials (Figure 3A). They swelled strongly upon placing in aqueous media (Figure 3B). Such swelling (~1.4-fold in the disc diameter) resulted in the pronounced softening of spongy matrices that absorbed, depending on the GA concentration, 12–14 ml of water per 1 g of the dry polymer (see Stv/w values in Table 1). Because of the softness of such water-swollen gelatin cryostructurates, the measurement of their mechanical characteristics gave low-informative results. Thus, when applying even a moderate load, squeezing of the capillary-bound liquid occurred, that complicated distinguishing the response of the proper gel phase (the walls of macropores) from the capillary forces. In turn, the swelling degree of the pore walls can be measured after squeezing the capillary liquid, weighing insoluble matter, its drying and final weighing (for the procedure see Section 2.2). Table 1 contains the data on Sw/w values for the gelatin cryostructurates prepared from the feed solutions with GA concentration 7.0, 8.5 and 10 wt.%. These values were about 4–5 times lower than the Stv/w values, thus indicating that the most fraction of the liquid inside the swollen GA-sponge corresponded to the capillary-bond water inside the space of large pores. In turn, the polymer content (24–25 wt.%) in the gel phase of swollen pore walls was 2–3-fold higher in comparison with the gelatin concentration in the feed solution (7–10 wt.%). It was the result of the freezing-induced concentrating effect typical for the cryostructuring processes in polymeric systems (27), (28), (29), (30), (58), (59). In general, the data of Table 1 testify that the cryostructurates formed in frozen DMSO solutions of gelatin and further cross-linked with EDC dissolved in ethanol, upon subsequent swelling in aqueous media “acquire” spongy morphology with large inner spaces of the gross pores. Such property provides the necessary conditions for the penetration of mammalian cells, when such matrices are employed as scaffolds for 3D cell culturing.

Figure 3: Discs of gelatin-based cryostructurates immersed in ethanol (A) or in water (B) (Petri dish’s inner diameter is 90 mm).
Figure 3:

Discs of gelatin-based cryostructurates immersed in ethanol (A) or in water (B) (Petri dish’s inner diameter is 90 mm).

Table 1:

Water-swelling parameters of gelatin cryostructurates.

GA concentration in feed solution (wt.%)Freezing temperature (°C)Total volumetric swelling degree of the gelatin sponge (Stv/w)a (ml H2O/g GA)Characteristics of gel phase within the gelatin sponge
Sw/wb (g H2O/g GA)GA content in the water-swollen pore walls (wt.%)
7.0−2014.9±1.53.24±0.1423.8±1.0
8.513.1±0.93.15±0.2124.3±1.7
10.012.0±1.03.09±0.1524.6±1.2

  1. aCalculated based on equation (1) (Section 2.2.2).

  2. bCalculated based on equation (2) (Section 2.2.2).

With that, certain specific features turned out to be inherent in the microstructure of gelatin cryostructurates prepared in accordance with the processing scheme given in Figure 2. The first of the features was the fact that macroporous morphology of the lower and top flat faces of the GA-discs was very different even for the sponges of 2-mm thickness. The micrographs in Figure 4 show the images of transmission light microscopy in a small magnification (A and B) and of laser confocal microscopy in a somewhat higher magnification (C and D). These pictures clearly demonstrate such structural differences. Thus, the honeycomb-like macroporous architecture was observed in the vicinity of bottom regions of the disc (Figure 4A and C), while the upper GA-sponge regions (Figure 4B and D) that contacted with air during freezing of the initial gelatin solution had rather irregular morphology. The cross-section of the pores observed within the lower face of the disc varied within the range of approximately 10–50 μm, while the size of the pores at the top face was much larger and varied within considerably wider limits (from ~50 to ~200 μm).

Figure 4: Micrographs of lower (A, C) and top (B, D) faces of the 2-mm-thick gelatin discs prepared originating from 7 wt.% GA dissolved in DMSO.(A and B) images taken with transmission optical microscope (staining with Amido Black dye); (C and D) images taken with laser confocal microscope (staining with Methylene Blue dye).
Figure 4:

Micrographs of lower (A, C) and top (B, D) faces of the 2-mm-thick gelatin discs prepared originating from 7 wt.% GA dissolved in DMSO.

(A and B) images taken with transmission optical microscope (staining with Amido Black dye); (C and D) images taken with laser confocal microscope (staining with Methylene Blue dye).

Similar morphology of the cryogenically structured gel matrices is known, for instance, for water-agarose systems (68), (69), when those were frozen in the molds placed onto the cooling plate. In such a case the grains of the solvent crystals are formed at the bottom areas of a sample, and further growth of crystals occurs along the temperature gradient direction. The porous morphology of the top regions in GA-discs (Figure 4B and D) testifies that DMSO crystals upon the growth in the viscous medium of gelatin solution quickly lost their initial vertical orientation and became “branched” thus resulting in the formation of rather disordered wide channels in the upper regions of the gel matrix. In any case, the pores inside these gelatin scaffolds were interconnected, and their size was large enough for the free penetration of cells to be seeded to the spongy scaffold.

3.3 Morphology, proliferation and metabolic activity of cells within 3D gelatin scaffolds

To evaluate the biocompatibility of wide porous gelatin matrices prepared, the MSCs were seeded into scaffolds and cultured in vitro. Following 24 h of 3D culture, confocal examination indicated good attachment of the MSCs to the scaffold walls on the surface and inside the construct. The degree of cell spreading on the top faces of the scaffolds was higher, compared to the lower faces, which was probably associated with the non-uniform microstructure of matrices, mentioned above. The flattened, fully-spread cell morphology was associated with the large and deep pores (Figure 5A), while the cell shape on the surface with the smaller pores varied from spherical to spindle-like (Figure 5B). According to FDA/EB staining, the cell survival did not depend on spreading, and the overwhelming majority of the MSCs were viable.

Figure 5: Morphology and viability of MSCs within the gelatin scaffold.(A) lower face of the bioconstruct; (B) top face (FDA/EB staining; green color – viable cells, red color – dead cells).
Figure 5:

Morphology and viability of MSCs within the gelatin scaffold.

(A) lower face of the bioconstruct; (B) top face (FDA/EB staining; green color – viable cells, red color – dead cells).

To find out whether gelatin scaffolds could support not only cell attachment but also cell growth we have studied the MSCs proliferation, by metabolic activity (AB) assay and determination of DNA content within 3D bio-constructs. The results showed a statistically significant increase in cell number by the end of the culture period (Figure 6A). The results of DNA analysis were confirmed by the AB assay of MSCs within the gelatin scaffolds. Figure 6B shows that the intensity of AB fluorescence on the 7th day of 3D culture increased by 32% compared to the 1st day of culture. Taking together, these results indicate that the MSCs were capable to attach to the pore surfaces of gelatin scaffolds, proliferate and distribute throughout the whole porous structure.

Figure 6: The quantitative data on the growth of MSCs within the gelatin scaffolds.(A) DNA content of MSCs cultured within gelatin scaffolds; (B) Alamar Blue assay of MSCs cultured within gelatin scaffolds. Data are presented as mean±SD. *Indicates significant differences.
Figure 6:

The quantitative data on the growth of MSCs within the gelatin scaffolds.

(A) DNA content of MSCs cultured within gelatin scaffolds; (B) Alamar Blue assay of MSCs cultured within gelatin scaffolds. Data are presented as mean±SD. *Indicates significant differences.

3.4 Differentiation of MSCs within gelatin scaffolds

Stem cell differentiation to a lineage of choice in a 3D environment can be crucial for achieving successful results when using tissue-engineered products for clinical application. In this study, we examined the ability of MSCs to differentiate towards the osteogenic and adipogenic lineages when cultured within the gelatin scaffolds.

ALP activity and calcium deposition were monitored to confirm osteogenic differentiation. To visualize the differentiation of mesenchymal cells to osteoblasts, ALP staining was performed after 3 weeks of culture in osteogenic media. Microscopic examination of the cell/scaffold bio-constructs revealed ALP expression, which is known to be an early marker of osteogenesis (Figure 7A). In the negative control (no osteoinductive media) MSCs continued to proliferate, formed almost continuous layer but were ALP negative.

Figure 7: Induced differentiation of MSCs within gelatin scaffolds (21 days of culture).(A) Osteogenic differentiation (alkaline phosphatase expression); (B) Adipogenic differentiation (lipid droplets are positively stained with Nile Red).
Figure 7:

Induced differentiation of MSCs within gelatin scaffolds (21 days of culture).

(A) Osteogenic differentiation (alkaline phosphatase expression); (B) Adipogenic differentiation (lipid droplets are positively stained with Nile Red).

After culture in adipogenic media for 3 weeks, the accumulation of intracellular lipid droplets stained with Nile Red was observed (Figure 7B). The differentiated cells were evenly distributed within the gelatin scaffolds and the extent of lipid droplets accumulation increased with prolongation of the period of induction. In contrast, the lipid droplets were not detected in scaffolds seeded with the MSCs and cultured in basic medium (control).

4 Conclusions

Diverse porous polymer materials are known to be used as scaffolds for 3D cell culture (16), (17). Such matrices possess a certain set of merits and demerits. The example of the latter case is the lack of sterility upon the scaffolds preparation, especially when the processes are accomplished in aqueous media. In order to minimize the hazard of possible microbial contamination we elaborated a new technique for the preparation of gelatin-based spongy scaffolds for 3D culturing of animal or human cells, particularly, multipotent MSCs. This preparation procedure includes sequential dissolution of Type A gelatin in dimethylsulfoxide, freezing such solution followed by the cryoextraction of crystalline phase with cold ethanol, cross-linking of biopolymeric chains with carbodiimide in ethanol medium, then treatment of the resultant spongy matrix with an ethanolic solution of Tris and final tanning of the material with formaldehyde also dissolved in ethanol. The use of organic media during all the preparation stages ensured sterility of the scaffolds, whose suitability for cell culturing was confirmed by providing the necessary environment for growth and differentiation of the multipotent MSCs. During the whole culture period (more than 3 weeks), the gelatin scaffolds did not change their integrity and strength, thus demonstrating good stability in the culture medium. Moreover, it is especially necessary to point out the absence of any microbial contamination of the GA-based sponges fabricated accordingly the procedure with the use of organic media, i.e. high sterility of the gelatin material was ensured. To sum up, the proposed novel technology for the sterile fabrication of 3D gelatin scaffolds provides opportunities for their wide application in the fields of tissue engineering and biotechnology.

Acknowledgments

The work on preparation of gelatin cryostructurates and examination of their properties was supported by the grant from the Russian Scientific Foundation; Project # 16-13-10-365. The biological study was carried out within the framework of the Program “Basic Research for Development of Biomedical Technologies” funded by the Presidium of Russian Academy of Sciences.

References

1. Veis A. The macromolecular chemistry of gelatin. New York: Academic Press; 1964.Search in Google Scholar

2. Djiagny VB, Wang Z, Xu S. Gelatin: a valuable protein for food and pharmaceutical industries: review. Crit Revs Food Sci Nutr. 2001;41:481–92.10.1080/20014091091904Search in Google Scholar PubMed

3. Jones RT. In: Pharmaceutical capsules. Podczeck F, Jones BE, editors. London: Pharmaceutical Press; 2004. 23–60 pp.Search in Google Scholar

4. Mouw JK, Ou G, Weaver VM. Extracellular matrix assembly: a multiscale deconstruction. Nat Rev Mol Cell Biol. 2014;15:771–85.10.1038/nrm3902Search in Google Scholar PubMed PubMed Central

5. Ellingsworth LR, Delustro F, Brennan JE, Sawamura S, McPherson J. The human immune-response to reconstituted bovine collagen. J Immunol. 1986;136:877–82.Search in Google Scholar

6. Lynn AK, Yannas IV, Bonfield W. Antigenicity and immunogenicity of collagen. J Biomed Mater Res. 2004;71B:343–54.10.1002/jbm.b.30096Search in Google Scholar PubMed

7. Chang CH, Kuo TF, Lin CC, Chou CH, Chen KH, Lin FH, Liu HC. Tissue engineering-based cartilage repair with allogenous chondrocytes and gelatin-chondroitin-hyaluronan tri-copolymer scaffold: a porcine model assessed at 18, 24, and 36 weeks. Biomaterials 2006;27:1876–88.10.1016/j.biomaterials.2005.10.014Search in Google Scholar PubMed

8. Gorgieva S, Girandon L, Kokol V. Mineralization potential of cellulose-nanofibrils reinforced gelatine scaffolds for promoted calcium deposition by mesenchymal stem cells. Mater Sci Eng Part C 2017;73:478–89.10.1016/j.msec.2016.12.092Search in Google Scholar PubMed

9. Hoque ME, Nuge T, Yeow TK, Nordin N, Prasad RGSV. Gelatin-based scaffold for tissue engineering – a review. Polym Res J. 2015;9:15–32.Search in Google Scholar

10. Kanda N, Anada T, Handa T, Kobayashi K, Ezoe Y, Takahashi T, Suzuki O. Orthotopic osteogenecity enhanced by a porous gelatin sponge in a critical-sized rat calvaria defect. Macromol Biosci. 2015;15:1647–55.10.1002/mabi.201500191Search in Google Scholar PubMed

11. Kemençe N, Bölgen N. Gelatin- and hydroxyapatite-based cryogels for bone tissue engineering: synthesis, characterization, in vitro and in vivo biocompatibility. J Tiss Eng Regen Med. 2017;11:20–33.10.1002/term.1813Search in Google Scholar PubMed

12. Zhao F, Grayson WL, Ma T, Bunnell B, Lu WW. Effects of hydroxyapatite in 3-D chitosan-gelatin polymer network on human mesenchymal stem cell construct development. Biomaterials 2006;27:1859–67.10.1016/j.biomaterials.2005.09.031Search in Google Scholar PubMed

13. Zuk P, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002;13:4279–95.10.1091/mbc.e02-02-0105Search in Google Scholar PubMed PubMed Central

14. Rogulska O, Petrenko Y, Petrenko A. DMSO-free cryopreservation of adipose-derived mesenchymal stromal cells: expansion medium affects post-thaw survival. Cytotechnology 2016;69:265–76.10.1007/s10616-016-0055-2Search in Google Scholar PubMed PubMed Central

15. Zonari A, Novikoff S, Electo NRP, Breyner NM, Gomes DA, Martins A, Neves NM, Reis RL, Goes AM. Endothelial differentiation of human stem cells seeded onto electrospun polyhydroxybutyrate/polyhydroxybutyrate-co-hydroxyvalerate fiber mesh. PLoS One. 2012;7:e35422.10.1371/journal.pone.0035422Search in Google Scholar PubMed PubMed Central

16. De Clerck E, De Vos P. Study of the bacterial load in a gelatine production process focussed on Bacillus and related endosporeforming genera. Syst Appl Microbiol. 2002;25:611–7.10.1078/07232020260517751Search in Google Scholar PubMed

17. De Clerck E, Gevers D, De Ridder K, De Vos P. Screening of bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis, focussed on Bacillus and related endospore-forming genera. J Appl Microbiol. 2004;96:1333–41.10.1111/j.1365-2672.2004.02250.xSearch in Google Scholar PubMed

18. Amadori S, Torricelli P, Rubini K, Fini M, Panzavolta S, Bigi A. Effect of sterilization and crosslinking on gelatin films. J Mater Sci Mater Med. 2015;26:69.10.1007/s10856-015-5396-4Search in Google Scholar PubMed

19. Islam MM, Khan MA, Rahman MM. Preparation of gelatin based porous biocomposite for bone tissue engineering and evaluation of gamma irradiation effect on its properties. Mater Sci Eng C Mater Biol Appl. 2015;49:648–55.10.1016/j.msec.2015.01.066Search in Google Scholar PubMed

20. Jagur-Grodzinski J. Polymers for tissue engineering, medical devices, and regenerative medicine. Concise general review of recent studies. Polym Adv Technol. 2006;17:395–418.10.1002/pat.729Search in Google Scholar

21. Shoichet MS. Polymer scaffolds for biomaterials applications. Macromolecules 2010;43:581–91.10.1021/ma901530rSearch in Google Scholar

22. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissues – state of the art and future perspectives. J Biomater Sci Polym Edn. 2012;12:107–24.10.1163/156856201744489Search in Google Scholar PubMed

23. Van Vlierberghe S. Crosslinking strategies for porous gelatin scaffolds. J Mater Sci. 2016;51:4349–57.10.1007/s10853-016-9747-4Search in Google Scholar

24. Blomeier H, Zhang X, Rives C, Brissova M, Hughes E, Baker M, Powers AC, Kaufman DB, Shear LD, Lowe WLJ. Polymer scaffolds as synthetic microenvironments for extrahepatic islet transplantation. Transplantation 2006;82:452–9.10.1097/01.tp.0000231708.19937.21Search in Google Scholar PubMed PubMed Central

25. Okay O. Macroporous copolymer networks. Progr Polym Sci. 2000;25:711–79.10.1016/S0079-6700(00)00015-0Search in Google Scholar

26. Van Vlierberghe S, Dubruel P, Schacht E. Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review. Biomacromolecules 2011;12:1387–408.10.1021/bm200083nSearch in Google Scholar PubMed

27. Lozinsky VI. Cryogels on the basis of natural and synthetic polymers: preparation, properties and areas of implementation. Russ Chem Revs. 2002;71:489–511.10.1070/RC2002v071n06ABEH000720Search in Google Scholar

28. Okay O, editor. Polymeric cryogels: macroporous gels with remarkable properties. Cham, Heidelberg, New-York, Dordrecht, London: Springer; 2014. 330 p.Search in Google Scholar

29. Lozinsky VI. A brief history of polymeric cryogels. Adv Polym Sci. 2014;263:1–48.10.1007/978-3-319-05846-7_1Search in Google Scholar

30. Kumar A, editor. Supermacroporous cryogels: biomedical and biotechnological applications. Boca Raton, FL: CRC Press, Taylor & Francis Group LLC; 2016. 480 p.10.1201/b19676Search in Google Scholar

31. Qian L, Zhang Y. Controlled freezing and freeze drying: a versatile route for porous and micro-/nano-structured materials. J Chem Technol Biotechnol. 2011;86:172–84.10.1002/jctb.2495Search in Google Scholar

32. Geidobler R, Winter G. Controlled ice nucleation in the field of freeze-drying: fundamentals and technology review. Eur J Pharm Biopharm. 2013;85:214–22.10.1016/j.ejpb.2013.04.014Search in Google Scholar PubMed

33. Barkay-Olami H, Zilberman M. Novel porous soy protein-based blend structures for biomedical applications: microstructure, mechanical, and physical properties. J Biomed Mater Res. 2016;104B:1109–20.10.1002/jbm.b.33459Search in Google Scholar PubMed

34. Rogozhin SV, Lozinsky VI, Vainerman ES, Domotenko LV, Mamtsis AM, Ivanova SA, Shtil’man MI, Korshak VV. Non-covalent cryostructurization in polymer systems. Doklady Akademii nauk SSSR. 1984;278:129–33. (in Russian).Search in Google Scholar

35. Van Vlierberghe S, Cnudde V, Dubruel P, Masschaele B, Cosijns A, De Paepe I, Jacobs PJS, Van Hoorebeke L, Remon JP, Schacht E. Porous gelatin hydrogels: 1. Cryogenic formation and structure analysis. Biomacromolecules 2007;8:331–7.10.1021/bm060684oSearch in Google Scholar PubMed

36. Dubruel P, Unger R, Van Vlierberghe S, Cnudde V, Jacobs PJS, Schacht E, Kirkpatrick CJ. Porous gelatin hydrogels: 2. In vitro cell interaction study. Biomacromolecules 2007;8:338–44.10.1021/bm0606869Search in Google Scholar PubMed

37. Van Vlierberghe S, Dubruel P, Lippens E, Masschaele B, Van Hoorebeke L, Cornelissen M, Unger R, Kirkpstrick CJ, Schacht E. Toward modulating the architecture of hydrogel scaffolds: curtains versus channels. J Mater Sci Mater Med. 2008;19:1459–66.10.1007/s10856-008-3375-8Search in Google Scholar PubMed

38. Jain E, Srivastava A, Kumar A. Macroporous interpenetrating cryogel network of poly(acrylonitrile) and gelatin for biomedical applications. J Mater Sci Mater Med. 2009;20:173–9.10.1007/s10856-008-3504-4Search in Google Scholar PubMed

39. Dainiak MB, Allan IU, Savina IN, Cornelio L, James ES, James SL, Mikhalovsky SV, Jungvid H, Galaev IY. Gelatin-fibrinogen cryogel dermal matrices for wound repair: preparation, optimization and in vitro study. Biomaterials 2010;31:67–76.10.1016/j.biomaterials.2009.09.029Search in Google Scholar PubMed

40. Van Vlierberghe S, Dubruel P, Schacht E. Effect of cryogenic treatment on the rheological properties of gelatin hydrogels. J Bioact Compat Polym. 2010;25:498–512.10.1177/0883911510377254Search in Google Scholar

41. Jurga M, Dainiak MB, Sarnowska A, Jablonska A, Tripathi A, Plieva FM, Savina IN, Strojek J, Jungvid H, Kumar A, Lukomska B, Domanska-Janik K, Fooraz N, McGuckin CP. He performance of laminin-containing cryogel scaffolds in neural tissue regeneration. Biomaterials 2011;32:3423–34.10.1016/j.biomaterials.2011.01.049Search in Google Scholar PubMed

42. Stancu IC, Dragusin DM, Vasile E, Trusca R, Antoniac I, Vasilescu DS. Prous calcium alginate-gelatin interpenetrating matrix and its biomineralization potential. J Mater Sci Mater Med. 2011;22:451–60.10.1007/s10856-011-4233-7Search in Google Scholar PubMed

43. Petrenko YA, Ivanov RV, Petrenko AY, Lozinsky VI. Coupling of gelatin to inner surfaces of pore walls in spongy alginate-based scaffolds facilitates the adhesion, growth and differentiation of human bone marrow mesenchymal stromal cells. J Mater Sci Mater Med. 2011;22:1529–40.10.1007/s10856-011-4323-6Search in Google Scholar PubMed

44. Elowsson L, Kirsebom H, Carmignac V, Durbeej M, Mattiasson B. Porous protein-based scaffolds prepared through freezing as potential scaffolds for tissue engineering. J Mater Sci Mater Med. 2012;23:2489–98.10.1007/s10856-012-4713-4Search in Google Scholar PubMed

45. Tripathi A, Vishnoi T, Singh D, Kumar A. Modulated crosslinking of macroporous polymeric cryogel affects in vitro cell adhesion and growth. Macromol Biosci. 2013;13:838–50.10.1002/mabi.201200398Search in Google Scholar PubMed

46. Inci I, Kirsebom H, Galaev IY, Mattiasson B, Piskin E. Gelatin cryogels crosslinked with oxidized dectran and containing freshly formed hydroxyapatite as potntial bone tissue-engineering scaffolds. J Tiss Eng Regen Med. 2013;7:584–8.10.1002/term.1464Search in Google Scholar PubMed

47. Shevchenko RV, Eeman M, Rowshanravan B, Allan IU, Savina IN, Illsey M, Salmon M, James SL, Mikhalovsky SV, James SE. The in vitro characterization of a gelatin scaffold, prepared by cryogelation and assessed in vivo as a dermal replacement in wound repair. Acta Biomater. 2014;10:3136–66.10.1016/j.actbio.2014.03.027Search in Google Scholar PubMed

48. Berillo D, Volkova N. Preparation and physicochemical characteristics of cryogel based on gelatin and oxidized dextran. J Mater Sci. 2014;49:4855–68.10.1007/s10853-014-8186-3Search in Google Scholar

49. Riva R, Omes C, Bassani R, Nappi RE, Mazzini G, Cornaglia AI, Casasco A. In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid. Reprod Biomed Online. 2014;29:457–69.10.1016/j.rbmo.2014.06.006Search in Google Scholar PubMed

50. Katsen-Globa A, Meiser I, Petrenko YA, Ivanov RV, Lozinsky VI, Zimmermann H, Petrenko AY. Towards a ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate-gelatin cryogel scaffolds. J Mater Sci Mater Med. 2014;25:857–71.10.1007/s10856-013-5108-xSearch in Google Scholar PubMed PubMed Central

51. Gorgieva S, Kokol V. Processing of gelatin-based cryogels with improved thermomechanical resistance, pore size gradient, and high potential for sustainable protein drug release. J Biomed Mater Res. 2015;103A:1119–30.10.1002/jbm.a.35261Search in Google Scholar PubMed

52. Lou CW, Wen SP, Lin JH. Chitosan/gelatin porous bone scaffolds made by crosslinking treatment and freeze-drying technology: effects of crosslinking durations on the porous structure, compressive strength, and in vitro cytotoxicity. J Appl Polym Sci. 2015;132:41851.10.1002/app.41851Search in Google Scholar

53. Nieto-Suárez M, López-Quintela MA, Lazzari M. Preparation and characterization of crosslinked chitosan/gelatin scaffolds by ice segregation induced self-assembly. Carbohydr Polym. 2016;141:175–83.10.1016/j.carbpol.2015.12.064Search in Google Scholar PubMed

54. Allan IU, Tolhurst BA, Shevchenko RV, Dainiak MB, Usley MI, Ivanov AE, Jungvid H, Galaev IY, James SL, Mikhalovsky SV, James SE. An in vitro evaluation of fibrinogen and gelatin containing cryogels as dermal regeneration scaffolds. Biomater Sci. 2016;4:1007–14.10.1039/C6BM00133ESearch in Google Scholar

55. Ferroni M, Siusti S, Nuscimento D, Silva A, Boschetti F, Ahluwalia A. Modelling the fluid dynamics and oxigen consumption in a porous scaffold stimulated by cyclic squeeze pressure. Med Eng Phys. 2016;38:725–32.10.1016/j.medengphy.2016.04.016Search in Google Scholar PubMed

56. Pandey C, Mittapelly N, Pant A, Sharma S, Singh P, Banala VT, Trivedi R, Shukla PK, Mishra PR. Dual functioning microspheres embedded crosslinked gelatin cryogels the therapeutic intervention in osteomyelitis and associated bone loss. Eur J Pharm Sci. 2016;91:105–13.10.1016/j.ejps.2016.06.008Search in Google Scholar PubMed

57. Lozinsky VI. New generation of macroporous and supermacroporous materials of biotechnological interest – polymeric cryogels. Russ Chem Bull. 2008;57:1015–32.10.1007/s11172-008-0131-7Search in Google Scholar

58. Lozinsky VI, Okay O. Basic principles of cryotropic gelation. Adv Polym Sci. 2014;263:49–101.10.1007/978-3-319-05846-7_2Search in Google Scholar

59. Okay O, Lozinsky VI. Synthesis, structure-property relationships of cryogels. Adv Polym Sci. 2014:263:103–57.10.1007/978-3-319-05846-7_3Search in Google Scholar

60. Rodionov IA, Grinberg NV, Burova TV, Grinberg VY, Shabatina TI, Lozinsky VI. Cryostructuring of polymer systems. 44. Freeze-dried and then chemically cross-linked wide porous cryostructurates based on serum albumin. e-Polymers 2017;17:263–74.10.1515/epoly-2016-0317Search in Google Scholar

61. Lozinsky VI, Kulakova VK, Petrenko AY, Petrenko YA, Ershov AG, Sukhanov YV. Composition for the preparation of macroporous carrier which is used upon 3D culturing of animal or human cells, and method for the fabrication of such carrier. Russian Patent. 2015;2,594,427.Search in Google Scholar

62. Petrenko YA, Ivanov RV, Lozinsky VI, Petrenko AY. Comparison of the methods for seeding human mesenchimal stem cells to macroporous alginate cryogel carriers. Bull Exp Biol Med. 2011;150:543–6.10.1007/s10517-011-1185-3Search in Google Scholar

63. Dankberg F, Persidsky MD. Test of granulocyte membrane integrity and phagocytic function. Cryobiology 1976;13:430–2.10.1016/0011-2240(76)90098-5Search in Google Scholar

64. Hammer Ø, Harper DAT, Ryan PD. Past: palentoological statisctics software package for education and data analysis. Palaeontol Electron. 2001;4:4.Search in Google Scholar

65. Romanova NA, Brovko LY, Ugarova NN. Comparative assessment of methods of intracellular ATP extraction from different types of microorganisms for bioluminescent determination of microbial cells. Appl Biochem Microbiol. 1997;33:306–11.Search in Google Scholar

66. Gordon AJ, Ford RA. Chemist’s companion. New York, NY: John Wiley and Sons; 1972.Search in Google Scholar

67. Tisato V, Cozzi E. Xenotransplantation: an overview of the field. Meth Mol Biol. 2012;885:1–16.10.1007/978-1-61779-845-0_1Search in Google Scholar PubMed

68. Yokoyama F, Achife EC, Momoda J, Shimamura R, Monobe K. Morphology of optically anisotropic agarose hydrogel prepared by directional freezing. Coll Polym Sci. 1990;268:552–8.10.1007/BF01410297Search in Google Scholar

69. Lozinsky VI, Damshkaln LG, Bloch KO, Vardi P, Grinberg NV, Burova TV, Grinberg VY. Cryostructuring of polymer systems. XXIX. Preparation and characterization of supermacroporous (spongy) agarose-based cryogels used as three-dimentional scaffolds for culturing insulin-producing cell aggregates. J Appl Polym Sci. 2008;108:3046–62.10.1002/app.27908Search in Google Scholar

Received: 2017-8-1
Accepted: 2017-8-22
Published Online: 2017-9-22
Published in Print: 2018-2-23

©2018 Walter de Gruyter GmbH, Berlin/Boston

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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https://doi.org/10.1515/epoly-2017-0151
Keywords for this article
3D culturing-differentiation; mesenchymal stromal cells; spongy scaffold; type A gelatin
Creative Commons
BY-NC-ND 3.0

Articles in the same Issue

  1. Frontmatter
  2. In this Issue
  3. Full length articles
  4. Poly(imide-siloxane)s based on hyperbranched polyimides
  5. Electrochemical, optical and morphological properties of poly (N-vinylcarbazole/TiO2) and (N-vinylcarbazole/aniline)/TiO2 copolymer prepared by electrochemical polymerization
  6. Synthesis and application of a novel core-shell-shell magnetic ion imprinted polymer as a selective adsorbent of trace amounts of silver ions
  7. New insights on solvent implications in flow behavior and interfacial interactions of hydroxypropylmethyl cellulose with cells/bacteria
  8. Electrochemical behavior, characterization and corrosion protection properties of poly(bithiophene+2-methylfuran) copolymer coatings on A304 stainless steel
  9. Fracture behavior of highly toughened poly(lactic acid)/ethylene-co-vinyl acetate blends
  10. Poly(N-isopropylacrylamide)-coated gold nanorods mediated by thiolated chitosan layer: thermo-pH responsiveness and optical properties
  11. Cryostructuring of polymer systems. 47. Preparation of wide porous gelatin-based cryostructurates in sterilizing organic media and assessment of the suitability of thus formed matrices as spongy scaffolds for 3D cell culturing
  12. Controlled drug delivery of ciprofloxacin from ultrasonic hydrogel
  13. Communication
  14. Synthesis and thermal properties of poly(vinylcyclohexane)-b-poly(4-vinylpyridine) diblock copolymers prepared via RAFT polymerization
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